Process for producing optically active 3-quinuclidinol derivatives

ABSTRACT

The present invention relates to a process for producing optically active 3-quinuclidinol or derivatives, wherein a racemic 3-quinuclidinol ester represented by the general formula (I): ##STR1## wherein R represents a straight-chain or branched alkyl group, and (H + ) represents that said ester may be in the form of a salt formed with a mineral acid or an organic acid, is reacted with a microorganism belonging to the genus Aspergillus, Rhizopus, Candida or Pseudomonas having the ability to asymmetrically hydrolyze said ester linkage, a culture of said microorganism, a treated material from said microorganism, an enzyme produced by said microorganism, or an enzyme derived from swine or cattle. 
     According to the present invention, there is provided a process for easily producing optically active 3-quinuclidinol derivatives which are important synthetic intermediates for pharmaceutical preparations etc.

TECHNICAL FIELD

The present invention relates to a process for producing opticallyactive 3-quinuclidinol derivatives which are very useful compounds asstarting materials or intermediates for pharmaceutical preparations,agrochemicals etc.

BACKGROUND ART

Processes for synthesizing racemic 3-quinuclidinol derivatives arewidely known according to e.g. Zhur. Obshchi. Khim., 30, 163-71 (1960),Helv. 40, 2107-85 (1957), and J. Am. Chem. Soc. 74, 2215-8 (1952).

On the other hand, processes for synthesizing optically active3-quinuclidinol derivatives are known as described in e.g. Acta. Pharm.Suec., 16, 281-3 (1979), U.S. Pat. No. 3,997,543, Life sic. 21,1293-1302 (1977) and U.S. Pat. No. 5,215,918.

The process described in Acta. Pharm. Suec., 16, 281-3 (1979) is aprocess in which optically active 3-quinuclidinol derivatives arederived by a preferential crystallization method using optically activetartaric acid as a resolution agent, and this prior art process requiresthe cumbersome procedure that e.g. recrystallization should be repeatedseveral or more times to raise optical purity.

Further, the processes described in U.S. Pat. No. 3,997,543, Life sic.21, 1293-1302 (1977) and U.S. Pat. No. 5,215,918 report processes forobtaining optically active 3-quinuclidinol derivatives by asymmetricallyhydrolyzing lower fatty esters of 3-quinuclidinol with an enzyme.However, the enzyme used in U.S. Pat. No. 3,997,543 and Life sic. 21,1293-1302 (1977) is a butyryl choline esterase only, and the enzyme usedin U.S. Pat. No. 5,215,918 is an only specific enzyme (subtilisin)produced by the genus Bacillus, and thus the type of enzyme used in anyof these literatures is limited so these cannot be said to be generalprocesses. There are still not known any other processes for producingoptically active 3-quinuclidinol derivatives by means of generalenzymes.

Accordingly, the useful process for synthesizing optically active3-quinuclidinol derivatives, provided by the present invention, has beendesired.

DISCLOSURE OF THE INVENTION

Accordingly, the object of the present invention is to provide a processfor easily producing optically active 3-quinuclidinol derivatives whichare useful intermediates and starting materials for optically activepharmaceutical preparations and optically active agrochemicals.

As a result of their eager study on the process for synthesizingoptically active 3-quinuclidinol derivatives, the present inventorsfound selected enzymes and microorganisms having an activity ofoptico-selectively hydrolyzing a racemic ester of 3-quinuclidinol, andthe present invention was thereby completed.

That is, the present invention relates to a process for producingoptically active 3-quinuclidinol or a salt thereof, wherein a racemic3-quinuclidinol ester represented by the general formula (I): ##STR2##wherein R represents a straight-chain or branched alkyl group, and (H⁺)represents that said ester may be in the form of a salt formed with amineral acid or an organic acid, is reacted with a microorganismbelonging to the genus Aspergillus, Rhizopus, Candida or Pseudomonashaving the ability to asymmetrically hydrolyze said ester linkage, aculture of said microorganism, a treated material from saidmicroorganism, an enzyme produced by said microorganism, or an enzymederived from swine or cattle.

Further, the present invention relates to a process for producingoptically active 3-quinuclidinol ester or a salt thereof, wherein aracemic 3-quinuclidinol ester represented by the general formula (I):##STR3## wherein R represents a straight-chain or branched alkyl group,and (H⁺) represents that said ester may be in the form of a salt formedwith a mineral acid or an organic acid, is reacted with a microorganismbelonging to the genus Aspergillus, Rhizopus, Candida or Pseudomonashaving the ability to asymmetrically hydrolyze said ester linkage, aculture of said microorganism, a treated material from saidmicroorganism, an enzyme produced by said microorganism, or an enzymederived from swine or cattle.

Hereinafter, the present invention is described in detail.

The straight-chain or branched alkyl group R in the starting material inthe process of the present invention, i.e. in the racemic ester of3-quinuclidinol represented by the general formula (I), is notparticularly limited insofar as it is an alkyl group containing 1 to 10carbon atoms, and specific examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, valel, isovalel, pival, hexyl,pentyl, octyl, decyl etc. Among those enumerated above, alkyl groupscontaining 1 to 5 carbon atoms are particularly preferable. Further,these may carry a substituted or unsubstituted phenyl group. As thesubstituent group, there may be mentioned an alkyl group, acyl group,halogen group and hydroxyl group.

The portion of the nitrogen atom in the racemic ester of 3-quinuclidinolmay intentionally have formed a salt with an organic acid or a mineralacid or the like as shown in the general formula (I). As the specificmineral acid salt, there may be mentioned hydrochloride, hydrobromate,sulfate, nitrate etc. As the organic acid salt, there may be mentionedaliphatic organic acid salts such as acetate, propionate, butyrate,fumarate, malonate and oxalate, as well as aromatic organic acid saltssuch as benzoate etc.

Hereinafter, the specific process of the present invention is described.

The starting material i.e. the racemic 3-quinuclidinol ester can beeasily synthesized in a method known in the art as described above. Thatis, racemic 3-quinuclidinol is first synthesized from isonicotinate,bromoacetate etc. as the starting material (J. Am. Chem. Soc. 74, 2215-8(1952)) and then reacted with an organic acid chloride or organic acidanhydride etc. whereby the desired ester can be synthesized.

The microorganisms belonging to the genera Aspergillus, Rhizopus,Candida and Pseudomonas producing an enzyme having the ability toproduce optically active 3-quinuclidinol and/or enantiomer estersthereof by asymmetric hydrolysis of the ester linkages in racemic3-quinuclidinol esters represented by the general formula (I) used inthe present invention are not particularly limited, and specificexamples include Aspergillus oryzae, Aspergillus melleus, Aspergillussojae, Aspergillus niger, Aspergillus saitoi, Candida antarctica andPseudomonas sp. MR2301 strain FERM BP4870.

A wide variety of lipases, proteases and esterases can be used as theenzyme produced by the microorganisms of the genera Aspergillus,Rhizopus, Candida and Pseudomonas capable of asymmetric hydrolysis usedin the present invention. Among these enzymes, those commerciallyavailable are e.g. Amano Seiyaku Lipase A6 (derived from the genusAspergillus), Amano Seiyaku Lipase AP4 (derived from the genusAspergillus), Amano Seiyaku Lipase AP6 (derived from the genusAspergillus), SIGMA Acylase I (derived from Aspergillus melleus), SIGMAProtease Type II (derived from Aspergillus oryzae), SIGMA Protease TypeXIII (derived from Aspergillus saitoi), SIGMA Protease (Newlase) TypeXVIII (derived from the genus Rhizopus), SIGMA Protease Type XIX(derived from Aspergillus sojae), SIGMA Protease Type XXIII (derivedfrom Aspergillus oryzae), Fluka Lipase (derived from Candidaantarctica), Nagase Seikagaku Kogyo Denazyme AP (derived fromAspergillus oryzae), Nagase Seikagaku Kogyo Denapsin 10P (derived fromAspergillus niger), NOVO Flavourzyme MG (derived from Aspergillusoryzae) etc.

Further, the enzyme derived from swine or cattle capable of asymmetrichydrolysis used in the present invention is not particularly limited,and it is possible to use a wide variety of lipases, proteases andesterases. The enzyme derived from swine or cattle includes SIGMAProtease Type I (derived from bovine spleen), SIGMA Acylase I (derivedfrom porcine kidney), SIGMA Trypsin (Type II) (derived from porcinespleen).

When the ester hydrolase described above is subjected to the reaction inthe process of the present invention, the mode of its use is notparticularly limited insofar as said enzyme exhibits the activity, andthe enzyme can be used not only in a purified form but also in a crudeform containing additives added for stabilizing the enzyme. Further, ifthe microorganism having the ability to produce the ester hydrolase asdescribed above is subjected to the reaction, the culture liquid itselfobtained by culturing the microorganism, or the microorganism harvestedfrom the culture by centrifugation etc., or the treated material can beused. If the enzyme is produced extracellularly, the culture afterremoving the microorganism by centrifugation can be used as such, but itis more effective to conduct the operation of concentration andpurification by ammonium sulfate treatment etc. The treated material ofthe microoganism includes a microorganism treated with acetone, tolueneetc., a lyophilized microorganism, a disrupted microorganism, acell-free extract from the disrupted microorganism, and a crude enzymesolution extracted from these materials. Further, the enzyme or themicroorganism can be immobilized for use so that their recovery andreuse can be facilitated after the reaction. For example, they can beimmobilized by inclusion into cross-linked acrylamide gel or physicallyor chemically immobilized on solid carriers such as ion-exchange resin,diatomaceous earth.

The enzyme derived from the microorganism used in the present inventionis obtained by culturing the microorganism producing said enzyme, andthe microorganism can be cultured in a liquid or solid medium. As thismedium, there is used a medium suitably containing ingredients such ascarbon sources, nitrogen sources, vitamins, minerals etc. which can beassimilated by said microorganism. To improve the hydrolysis ability ofthe microorganism, a small amount of inducers etc. can also be added tothe medium. Culture is performed at the temperature and pH at which themicroorganism can grow, preferably under the optimum culture conditionsfor the strain used. To promote growth of the microorganism, stirringunder aeration is conducted in some cases.

For optico-selective hydrolysis of the racemic 3-quinuclidinol ester,the racemic 3-quinuclidinol ester serving as the substrate is dissolvedor suspended in a reaction solvent. Then, the enzyme, the microorganism,the culture of the microorganism or the treated material from themicroorganism, which act as a catalyst, is added thereto, and whilecontrolling the reaction temperature and the pH of the reaction solutionif necessary, the reaction is continued until approximately half of the3-quinuclidinol esters are hydrolyzed. Depending on the case, thereaction may be terminated at an initial stage or may proceedexcessively.

The concentration of the substrate during the reaction is notparticularly limited in the range of 0.1 to 70% by weight, but inconsideration of the solubility, productivity etc. of the racemic3-quinuclidinol ester as the substrate, the reaction is conducted usingthe substrate preferably in the range of 5 to 50% by weight. Thesubstrate can be added thereto as such or in the form of a solution inan organic solvent.

As described above, the portion of the nitrogen atom in the3-quinuclidinol derivative may have formed a salt with an organic acidor a mineral acid or the like.

As regards the reaction time, conditions are selected such that thereaction is finished usually for 1 hour to 1 week, preferably for 1 to72 hours.

The reaction pH depends on the optimum pH of the microbial enzyme used,but it is preferable that the reaction is conducted usually in the rangeof pH 4 to 11, particularly in the range of pH 6 to 9.5 in order toprevent the reduction of optical purity caused by chemical hydrolysis.As the reaction proceeds, the pH of the reaction solution is decreaseddue to the carboxylic acid formed, and in this case, the progress of thereaction is often promoted by maintaining the optimum pH with a suitableneutralizing agent such as sodium hydroxide, potassium hydroxide etc.The reaction temperature is preferably in the range of 5 to 70° C., morepreferably 10 to 60° C.

Usually, the reaction solvent makes use of aqueous media such asdeionized water, buffers etc., but the reaction can also be effectedeven in a system containing an organic solvent. For example, it ispossible to suitably use organic solvents e.g. alcohols such asmethanol, ethanol, propanol, isopropanol, butanol, isobutanol,tert-butanol, tert-amyl alcohol etc., ethers such as diethyl ether,isopropyl ether, dioxane etc., ketones such as acetone, methyl ethylketone, methyl isobutyl ketone etc., aromatic and hydrocarbon solventssuch as hexane, octane, benzene, toluene etc. The reaction can also beeffected in a 2-phase system in which the organic solvent is added insuch excess as not to be dissolved in water.

There are many cases where selectivity, transformation, yield etc. areimproved by allowing the organic solvent to be present in the reactionsystem.

The substrate concentration, reaction temperature, reaction solvent,reaction time, oxygen concentration and other reaction conditionsdescribed above are suitably selected in consideration of the reactionyield, optical yield etc. under such conditions, in order to attain themaximum amount of the desired optically specific compounds to berecovered.

According to the reaction described above, a reaction solutioncontaining optically active 3-quinuclidinol whose ester linkage wasasymmetrically hydrolyzed and its enantiomer (unreacted enantiomerester), that is, the optically active 3-quinuclidinol ester which wasnot hydrolyzed, can be obtained from the racemic 3-quinuclidinol ester.

Isolation of the optically active 3-quinuclidinol or the opticallyactive 3-quinuclidinol ester (unreacted enantiomer ester) from theresulting reaction solution can make use of any separation techniquessuch as distillation, extraction, separation on columns,recrystallization etc. known in the art.

The optically active 3-quinuclidinol ester (unreacted enantiomer ester)can be obtained for example by removing the enzyme or the microorganismused as the catalyst by operations such as centrifugation, filtrationetc., then adjusting the pH, and extracting it with a solvent such ashexane, chloroform or ethyl acetate.

On the other hand, the hydrolyzed optically active 3-quinuclidinol canbe obtained for example by concentrating the extracted residue andfurther re-crystallizing it from a solvent such as benzene, toluene,acetone etc.

Prior to separation of the optically active 3-quinuclidinol from itsenantiomer ester, a part or the whole of the reaction solvent may beremoved by distillation etc. depending on the amount and type of theorganic solvent contained in the reaction solvent, and then a suitableorganic solvent may be added thereto depending on the case.

The resulting optically active 3-quinuclidinol ester (unreactedenantiomer ester) can be hydrolyzed in a usual manner whereby the esterwhile maintaining the optical activity can be converted into3-quinuclidinol. Further, the optically active 3-quinuclidinol can beesterified in a usual manner whereby the 3-quinuclidinol whilemaintaining the optical activity can be converted into the3-quinuclidinol ester. Accordingly, the optically active 3-quinuclidinolderivatives with arbitrary configuration can be obtained.

These optically active 3-quinuclidinol derivatives contain tertiaryamines, so their amine salts can be formed in a usual manner.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in more detail byreference to Reference Examples and typical Examples, but these examplesare not intended to limit the scope of the present invention. [ReferenceExample 1] Synthesis of racemic 3-quinuclidinol ester.

1) Synthesis of 3-quinuclidinyl acetate

8.1 g of acetic anhydride was dropped slowly to 10 g of 3-quinuclidinol(Tokyo Kasei Kogyo K. K.), and the mixture was stirred for 20 hours atroom temperature to synthesize 3-quinuclidinyl acetate.

2) Synthesis of 3-quinuclidinyl butyrate

12.5 g of butyric anhydride was dropped slowly to 10 g of3-quinuclidinol, and the mixture was stirred for 20 hours at roomtemperature to synthesize 3-quinuclidinyl butyrate.

EXAMPLE 1

10.0 g of 3-quinuclidinyl butyrate was dissolved in 150 ml of 50 mMphosphate buffer (pH 7), then 5.0 g of Denazyme AP from Nagase SeikagakuKogyo was added thereto, and the reaction was initiated at 30° C. whilemaintaining pH 7.0 with 0.5 N NaOH. When the approximately theoreticalamount of the alkali was added, the reaction was terminated, and hexanewas added thereto, and sodium carbonate was added thereto understirring, thus making the pH of the aqueous phase alkaline and theliquid was separated. The hexane layer was concentrated to give 2.5 g of(R) 3-quinuclidinyl butyrate. Specific rotation [α]²⁴ _(D) =+34 (neat).

The resulting (R) 3-quinuclidinyl butyrate was hydrolyzed withwater-NaOH and then recrystallized from toluene to give(R)-quinuclidinol. Specific rotation [α]²⁴ _(D) =-45 (1N HCl).

The resulting (R) 3-quinuclidinyl butyrate was dissolved in a 5-foldexcess volume of methanol, and 3-fold molar equivalents of conc.hydrochloric acid were added thereto and the mixture was refluxed for 5hours. The reaction solution was concentrated into dryness to give (R)3-quinuclidinol hydrochloride quantitatively.

The (S) 3-quinuclidinol hydrolyzed by the enzyme reaction was recoveredby removing water and the enzyme followed by recrystallization fromtoluene in a similar manner. Specific rotation [α]²⁴ _(D) =+37 (1N HCl).

EXAMPLE 2

3-Quinuclidinyl butyrate was dissolved at 5% by weight in 500 mMphosphate buffer (pH 7), and an enzyme shown in Table 2 was addedthereto suitably in the range of 1 to 5% by weight and allowed to reactat 30° C. for 20 hours. The concentration and optical purity of the3-quinuclidinol were determined by gas chromatography (column:CP-Chirasil DEX CB 0.25×25 M, from Chrom Back). The results arecollectively shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Results of Asymmetric Hydrolysis of 3-Quinuclidinyl Butyrate                                       concentration of                                                                          optical purity of                              Enzymes 3-quinuclidinol 3-quinuclidinol                                     ______________________________________                                        SIGMA Acylase I: 1.0 wt-%    100% ee (S)-body                                   Aspergillus melleus                                                           SIGMA Protease Type I: 1.1 wt-%  55% ee (S)-body                              Bovine Pancreas                                                               SIGMA Protease Type II: 1.1 wt-%  80% ee (S)-body                             Aspergillus oryzae                                                            SIGMA Protease (Newlase) Type 0.8 wt-% 100% ee (S)-body                       XVIII: Rhizopus species                                                       SIGMA Protease Type XIX: 0.9 wt-%  80% ee (S)-body                            Aspergillus sojae                                                             SIGMA Protease Type XXIII: 1.6 wt-%  29% ee (S)-body                          Aspergillus oryzae                                                            SIGMA Trypsin Type II: 1.5 wt-%  30% ee (S)-body                              Porcine Pancreas                                                              Fluka Lipase: Candida antarctica 1.6 wt-%  22% ee (S)-body                    NOVO Flavourzyme MG: 1.1 wt-%  70% ee (S)-body                                Aspergillus oryzae                                                          ______________________________________                                    

EXAMPLE 3

3-Quinuclidinyl acetate was dissolved at 5% by weight in 500 mMphosphate buffer (pH 7), and an enzyme shown in Table 2 was addedthereto suitably in the range of 1 to 5% by weight and allowed to reactat 30° C. for 20 hours. The concentration and optical purity of the3-quinuclidinol were determined by gas chromatography (column:CP-Chirasil DEX CB 0.25×25 M, from Chrom Back). The results arecollectively shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Results of Asymmetric Hydrolysis of 3-Quinuclidinyl Acetate                                        concentration of                                                                          optical purity of                              Enzymes 3-quinuclidinol 3-quinuclidinol                                     ______________________________________                                        Amano Seiyaku Lipase A6                                                                        2.1 weight-%                                                                               16% ee (S)-body                                   Amano Seiyaku Lipase AP4 1.8 weight-%  23% ee (S)-body                        Amano Seiyaku Lipase AP6 2.5 weight-%  13% ee (S)-body                        SIGMA Acylase I: Porcine Kidney 0.9 weight-%  75% ee (S)-body                 SIGMA Protease Type II: 0.5 weight-% 100% ee (S)-body                         Aspergillus oryzae                                                            SIGMA Protease Type XIII: 0.8 weight-%  51% ee (S)-body                       Aspergillus saitoi                                                            SIGMA Protease Type XXIII: 0.9 weight-%  75% ee (S)-body                      Aspergillus oryzae                                                            Nagase Seikagaku Kogyo 0.7 weight-%  65% ee (S)-body                          Denazyme AP: Aspergillus oryzae                                               Nagase Seikagaku Kogyo 0.7 weight-%  50% ee (S)-body                          Denapsin 10P: Aspergillus niger                                             ______________________________________                                    

EXAMPLE 4

Pseudomonas sp. MR2301 strain FERM BP4870 was inoculated into 50 ml LBmedium (1 weight-% polypeptone, 0.5 weight-% yeast extract, 0.5 weight-%NaCl) containing 0.1 weight-% lactamide and cultured at 30° C. for 48hours with shaking. After culturing was finished, the culture wascentrifuged, and the whole of the microorganism thus obtained was washedwith deionized water and suspended in 50 ml of 500 mM phosphate buffer(pH 7.0). 2.5 g of racemic 3-quinuclidinyl acetate was added to thismicrobial suspension and allowed to react at 30° C. for 20 hours. Thepresence of 3-quinuclidinol at a concentration of 1.3 weight-% with anoptical purity of 31% ee (S)-body was confirmed by analyzing thereaction solution by gas chromatography (column: CP-Chirasil DEX CB,from Chrom Back).

Industrial Applicability

According to the present invention, there is provided a process foreasily producing optically active 3-quinuclidinol derivatives which areimportant synthetic intermediates for pharmaceutical preparations etc.

What is claimed is:
 1. A process for producing optically active3-quinuclidinol or a salt thereof, wherein a racemic 3-quinuclidinolester represented by the general formula (I): ##STR4## wherein Rrepresents a straight-chain or branched alkyl group containing 1 to 10carbon atoms, and (H+) represents that said ester may be in the form ofa salt formed with a mineral acid or an organic acid, is reacted with amicroorganism having the ability to asymmetrically hydrolyze said esterlinkage, wherein said microorganism is selected from the groupconsisting of Rhizopus sp., Aspergillus oryzae, Aspergillus melleus,Aspergillus sojae, Aspergillus niger, Aspergillus saitoi, Candidaantarctica, and Pseudomonas sp. MR2301 strain FERM BP4870, a culture ofsaid microorganism, a treated material from said microorganism, or anenzyme produced by said microorganism, wherein said treated materialfrom said microorganism is selected from the group consisting of themicroorganism treated with acetone, the microorganism treated withtoluene, the lyophilized microorganism, the disrupted microorganism, acell-free extract from the microorganism, and a crude enzyme solutionextracted from the microorganism, and wherein said enzyme is a lipase,protease or esterase.
 2. A process for producing optically active3-quinuclidinol or a salt thereof, wherein a racemic 3-quinuclidinolester represented by the general formula (I): ##STR5## wherein Rrepresents a straight-chain or branched alkyl group containing 1 to 10carbon atoms, and (H⁺) represents that said ester may be in the form ofa salt formed with a mineral acid or an organic acid, and is reactedwith a protease, acylase or trypsin derived from swine or cattle havingthe ability to asymmetrically hydrolyze said ester linkage.
 3. A processfor producing optically active 3-quinuclidinol ester or a salt thereof,wherein a racemic 3-quinuclidinol ester represented by the generalformula (I): ##STR6## wherein R represents a straight-chain or branchedalkyl group containing 1 to 10 carbon atoms, and (H⁺) represents thatsaid ester may be in the form of a salt formed with a mineral acid or anorganic acid, is reacted with a microorganism having the ability toasymmetrically hydrolyze said ester linkage, wherein said microorganismis selected from the group consisting of Rhizopus sp., Aspergillusoryzae, Aspergillus melleus, Aspergillus sojae, Aspergillus niger,Aspergillus saitoi, Candida antarctica, and Pseudomonas sp. MR2301strain FERM BP4870, a culture of said microorganism, a treated materialfrom said microorganism, or an enzyme produced by said microorganism,wherein said treated material from said microorganism is selected fromthe group consisting of the microorganism treated with acetone, themicroorganism treated with toluene, the lyophilized microorganism, thedisrupted microorganism, a cell-free extract from the microorganism, anda crude enzyme solution extracted from the microorganism, and whereinsaid enzyme is a lipase, protease or esterase.
 4. A process forproducing optically active 3-quinuclidinol ester or a salt thereof,wherein a racemic 3-quinuclidinol ester represented by the generalformula (I): ##STR7## wherein R represents a straight-chain or branchedalkyl group containing 1 to 10 carbon atoms, and (H⁺) represents thatsaid ester may be in the form of a salt formed with a mineral acid or anorganic acid, and is reacted with a protease, acylase or trypsin derivedfrom swine or cattle having the ability to asymmetrically hydrolyze saidester linkage.